CN1146657C

CN1146657C - Construction and use of recombinant herpes simplex virus

Info

Publication number: CN1146657C
Application number: CNB011005793A
Authority: CN
Inventors: 吴小兵; 曹晖; 侯云德
Original assignee: AGTC Gene Technology Co Ltd
Current assignee: AGTC Gene Technology Co Ltd
Priority date: 2001-01-16
Filing date: 2001-01-16
Publication date: 2004-04-21
Anticipated expiration: 2021-01-16
Also published as: CN1299868A

Abstract

The present invention provides a recombined herpes simplex virus whose natural packaging signal (pac) is deficient, a reformed packaging signal replaces the natural packaging signal (pac), wherein both ends of the packaging signal respectively have a recombined sequence (a loxP sequence), and the other point in a recombined virus genome is inserted with a code specially identifying a gene of the recombinase Cre of the recombined sequence. The Cre gene has an adjustable promoter (a metallothionein promoter) to control Cre expression. After the recombined virus infects a cell, a proper quantity of zinc ions are added to activate the metallothionein promoter, the recombinase Cre is expressed, recombination is generated between two loxP sequences mediated by the Cre, and the packaging signal is cut. Thereby, although the virus genome can copy and express various proteins, the virus genome can not be packed into a virus capsid, and therefore, a progeny virus is not generated. If the cell is not added with the zinc ions, the Cre is not expressed, the packaging signal on the recombined virus genome is not cut, and therefore, the recombined virus can be normally copied and carries out passage in the cell. The recombined virus provided by the present invention can be used as a helper virus of an amplicon carrier of the herpes simplex virus and is directly used as a gene treating carrier to be used for the treatment of malignant tumors and the preparation of vacant shells of the virus.

Description

A kind of structure of recombinant herpes simplex virus and purposes

Invention field

The invention belongs to the biotechnology invention field.Be specifically related to a kind of recombinant herpes simplex virus (Herpes Simplex Virus, structure HSV) and purposes.The recombinant herpes simplex virus that the present invention proposes is a kind of " the HSV recombinant virus that contains controlled removal packaging signal "

Background technology hsv (herpes simplex virus) belongs to herpetoviridae, Alphaherpesvirinae, and representative strains is herpes simplex types 1 virus (HSV-1).HSV-1 can infect the cell of nearly all type, especially has the characteristics of having a liking for neurocyte, can be through the cynapse direct motion or the antidromic propagation of neurocyte, and can in neurone, set up stable inapparent infection and the function of the cell that do not affect the nerves.Therefore HSV-1 is expected as a kind of new virus carrier that foreign gene is imported body cell, especially neurocyte.

The HSV-1 genome is that a molecular weight is the double-stranded linear DNA of 152kb, and it is by forming by covalently bound long segment (L) and short-movie section (S).L and S account for 82% and 18% of whole genome respectively, and inverted repeats (inverted repeat) is all contained at the two ends of L and S, is present in genomic inside (IR respectively _L, IR _S) and terminal (TR _L, TR _S), the centre is unique UL and US sequence.The reverse repetition at L fragment two ends is with ab and b ' a ' expression, and S fragment two ends are represented with a ' c ' and ca.Usually in the L-S joining region and the terminal a sequence of L fragment multiple copy number be different, but have only a of single copy usually at S fragment end.The HSV-1 genome of standard can be expressed as a _mB-U _L-b ' a ' _mC '-Us-ca, n and m do not wait from 1-10.Comprise virus genomic cutting and packaging signal among a, behind the HSV cells infected, its wire genome is closed into ring-type, and carries out rolling-circle replication in nucleus, produces end to end big DNA chain, is called mosaic (concatemer).Mosaic is discerned by viral capsid proteins at a place, is cut into the DNA of viral genome size, and packing is advanced in the capsid.A sequence size in different HSV-1 strains does not wait from 280-550bp.It comprises a plurality of (DR) and special (U) sequences that repeat in the same way.Such as, in HSV-1 (F) virus strain, a sequential structure is DR1-U _C-(DR4) _X-(DR2) _Y-U _b-DR1, DR1 wherein, DR2 and DR4 respectively long 20,12 and 37bp, Uc and Ub long 58 and 64bp.The genomic chimeric cutting of HSV-1 occurs in two DR1 places between a copy.Each only contains the part of 20bpDR1 the segmental free-end of L and S, and wherein the segmental terminal a sequence of L comprises 18bp and one 3 ' overhang, and the segmental terminal a sequence of S only has 1bp and one 3 ' overhang.When the viral DNA cyclisation of wire two strands, two terminal common complete DR1 that constitute thus.Therefore from the viral genome angle of cyclisation, two zones that contain a are arranged, on it hereinafter to be referred as pac (package).These two pac lay respectively at virus genomic two L and the segmental junction of S, and they are virus genomic packaging signals.Studies show that a pac just can finish virus genomic cutting and packaging function, if but two pac lack, the mosaic that viral genome produces in cell can't further be cut and be packed in the viral capsid, thereby can't produce virus of future generation.

The reorganization HSV that summary of the invention is proposed by the invention hereinafter to be referred as rHSV/LaL-MtCre, is characterized in that following 3 points: original two packaging signal pac disappearance in (1) HSV-1 genome; (2) insert in genomic a certain site as the packaging signal through transforming, these packaging signal both sides respectively have a loxP sequence of arranging in the same way.The loxP sequence is the specific recognition site of recombinase Cre.(3) inserted a gene cre who expresses Cre in virus genomic another site, this upstream region of gene is a metallothionein(MT) (metallothioneine, Mt) promotor, this promotor just start the cre expression of gene when only zinc ion concentration reaches certain level in environment.

Except that the phage P1 Cre/loxP system that the present invention uses, other similar DNA recombinase system, such as (the Mark et al.Nucleic Acids Research of yeast Flp/Frt system, 20,4451-4455 (1992)) and R/Ft (Kilby et al.1993, TIG9 413-420) also can be used for the present invention.Except that the metallothionein promoter that the present invention proposes, regulatable promotor can also be other type: hormone (adrenocortical hormone glucocorticoids GRE, Progesterone progesterones PRE, oestrogenic hormon estrogens ERE etc.), the viral protein (promotor that contains TRA or RRE is corresponding to be activated by the TAT of HIV or REV albumen) or the regulation and control substance (containing the UAS-Gal4 sequence activated by Gal4) in cell source, or bacterium tsiklomitsin promotor (being subjected to the activation of tTA) etc.

Recombinase Cre is phage-coded 343 amino acid whose polypeptide chains, and it can make two loxP sequences (34 base pair) of arranging in the same way recombinate specifically, thereby causes the disappearance of DNA therebetween.The Cre recombinase acts on loxP, does not rely on duplicating and external source energy (such as ATP) of DNA, and protokaryon or eucaryon, superhelix or linear DNA are all worked.(Sternberg，N.，Hamilton，D.，J.Mol.Biol.1981，150，467-486)。Among the constructed rHSV/LaL-MtCre of the present invention, loxP-pac-loxP is inserted in HSV-1 UL44 gene (dispensable gene, coding viral glycoprotein C) in the XbaI site, the MtCre gene is inserted in the XbaI site of HSV-1 UL2 gene (dispensable gene, coding viruria pyrimidine-DNA glycosylase) (accompanying drawing 1 is the design of graphics of recombinant virus rHSV/LaL-MtCre).Advance the viral genome fragment generation homologous recombination on 5 clays of cell through transfection, form the cyclic viral genome.The latter carries out rolling-circle replication and forms connected head-to-tail mosaic, expresses the early, middle and late phase albumen of virus simultaneously, and mosaic is cut into wire genome about 152kb.Because chimeric cutting occurs in the inside of a among the pac, so pac is cut into two sections, lays respectively at genomic two ends of rHSV/LaL-MtCre wire, forms ' the structure of pac-loxP-genome-loxP-psc '.Behind the rHSV/LaL-MtCre cells infected, the pac at these genome two ends links to each other, and forms the ring-type genome, carries out virus genomicly duplicating and packing.

When rHSV/LaL-MtCre infected common HSV-1 sensitive cells, the Cre recombinase was not expressed, rHSV/LaL-MtCre energy normal replication and amplification; When containing an amount of zine ion in the cell that rHSV/LaL-MtCre infects, the Mt promotor is activated, cre genetic expression Cre recombinase, Cre excises the loxP-pac-loxP of rHSV/LaL-MtCre specifically, place, point of contact viral DNA is from being dynamically connected, leave to loopholes, so rHSV/LaL-MtCre virus can't pack out progeny virus, but viral genome still can duplicate in cell and express.Before the present invention, we have made up strain reorganization HSV virus: rHSV/LaL (Cao Hui etc., the virus journal, 2000,16 (4): 374-377) (Chinese patent application number: 99122067.6), this virus contains loxP-pac-loxP, but does not contain regulatable Cre gene, so its packaging signal is to excise by the Cre albumen that trans (as cell strain) expresses.

The rHSV/LaL-MtCre that the present invention proposes is by a cover being contained complete genomic clay plasmid (the SetC clay: comprise cos6 of HSV-1 virus, cos14, cos28, cos48, cos56) produce on the basis that (Cunningham C.and Davison A.J.1993, Virology 197:116-124) transforms, at first, original two the packaging signal pac excision of HSV-1 genome on clay plasmid cos6 and the cos48 be will be arranged in, cos6 Δ a and cos48 Δ a become.Then, the pac that both sides have been loaded onto in the same way the Cre recombinase specific recognition site loxP that arranges inserts in the XbaI site of dispensable gene UL44 of the HSV-1 on the cos56, becomes cos56/loxP-pac-loxP; The cre gene that will have metallothionein promoter inserts in the XbaI site of cos6 Δ a, becomes cos6 Δ a-MtCre.With cos6 Δ a-MtCre, cos56/loxP-pac-loxP, cos28, cos14, five clays of cos48 Δ a after the PacI enzyme cuts clay skeleton part, with liposome method cotransfection HSV-1 sensitive cells (as BHK-21), homologous recombination takes place and produces recombinant virus rHSV/LaL-MtCre in 5 HSV-1 fragments in cell.This recombinant virus can be stablized in not containing the BHK-21 cell of zine ion and goes down to posterity.In containing the BHK-21 cell of an amount of zine ion, can not or only produce a small amount of progeny virus.

The rHSV/LaL-MtCre that the present invention proposes can further transform, to adapt to different requirements.Such as, the necessary gene of some virus infection (as envelope protein gH) on its genome is removed, the latter provides by cell strain is trans, to reach the purpose of the auxilliary poison of further minimizing.If γ 34.5 genes such as grade of HSV-1 are removed, can make its infection somatoblast, such as tumour cell, and do not infect Unseparated Cell, normal cell such as human body, therefore except as helper virus, it also is expected to directly as transgenosis and Vectors in Gene Therapy, can realize control to the amount of this recombinant virus by the concentration that changes the zine ion in the environment.

The rHSV/LaL-MtCre that the present invention proposes has the purposes of following several respects:

(1) as the helper virus of packaging virus carrier

Virus vector plays important effect in transgenosis and gene therapy, HSV-1 can be used for the auxiliary package of multiple virus vector as helper virus.

1) as the helper virus of HSV-1 amplicon (amplicon) carrier.HSV-1 can infect the cell of nearly all type, especially has the characteristics of having a liking for neurocyte, can be through the cynapse direct motion or the antidromic propagation of neurocyte, and can in neurone, set up stable inapparent infection and the function of the cell that do not affect the nerves.Therefore, the virus vector based on HSV-1 is expected as a kind of new virus carrier that foreign gene is imported body cell, especially neurocyte.The HSV-1 amplicon vector only contains HSV-1 virus replication and the necessary cis element of packing, and promptly replicon oris and packaging signal pac also comprise the foreign gene and protokaryon replicon and the resistant gene that need transfer in addition.The required trans element of amplicon virus packing is usually by helper virus HSV-1 or contain auxiliary package system such as virus genomic plasmid DNA and provide.Therefore amplicon virus is the damaged virion of a kind of height, and it does not contain the structure gene of HSV virus, has good security, and the bale capacity of foreign gene big (greater than 30kb, can reach nearly 150kb in theory).In the cell that trans element can be provided, the HSV-1 amplicon vector plasmid that enters cell with rotaring transfecting mode duplicates the connected head-to-tail mosaic of generation through " rolling ring ", is cut then, is packaged into and contains the pseudovirus that multiple copied foreign gene, DNA always are about 150kb.Therefore the HSV-1 amplicon vector has higher exogenous gene expression amount.For example, the amplicon plasmid of a 10kb size is packaged into the amplicon virus of a 150kb, wherein contains 15 copies, end to end this amplicon plasmid.With a few viroid carriers commonly used at present, compare such as retroviral vector, adenovirus carrier, gland relevant viral vector, the advantage of HSV-1 amplicon virus vector is: carrier contains the foreign gene of a plurality of copies, advantages such as expression of exogenous gene amount height, bale capacity big (can pack greatly the exogenous genetic fragment of 15～30kb), vector construction are simple, thereby be subjected to more concern day by day in transgenosis and field of gene.The HSV-1 amplicon vector has been widely used in the research and clinical preceding application of transgenosis and gene therapy, and has obtained significant progress.But, often containing a large amount of HSV-1 helper viruses in the amplicon vector, HSV-1 has cytotoxicity.Make amplicon vector enter clinical experiment, must reduce until removing helper virus fully.HSV-1 amplicon vector auxiliary package system commonly used at present has following two classes usually: a class is as helper virus (Geller with HSV-1 replication defect type mutant strain, A.L, K.Keyomarski, J.Bryan, A.B.Pardee.1990.Proc.Natl. Acad.Sci.USA 87:8950-8954).This class helper virus has plenty of the necessary gene of certain virus replication of disappearance, and the albumen of this genes encoding provides by cell is trans; Have plenty of temperature sensitive mutant, can normal replication during 31 ℃ of this helper viruses, and reproducible not 37 ℃ the time.Contain a large amount of helper viruses in the amplicon virus that this method obtains,, but still infection ability is arranged though these helper viruses can't normal replication.This type of helper virus has following problem: the 1. cytotoxicity and the immune response that bring of the genetic expression of helper virus: 2. potential interacts between helper virus and endogenous contaminating virus: 3. amplicon virus brings the instability of foreign gene to express in the hybrid virus with the fluctuation of helper virus ratio; 4. helper virus potential carcinogenesis: 5. helper virus reverts back to the possibility of wild-type virus.Another kind of is the auxilliary malicious packaging system of nothing of amplicon virus vector, the HSV-1 genome that normally will lack packaging signal is placed on five clay (Fraefel C, Song S, Lim F, et al.J virology, 1996, (Stavropoulos TA 70:7190-7197) or in the bacterial artificial chromosome, Strathdee CA.Virology, 1998,72:7137～7143), by with amplicon plasmid co-transfection mode transfered cell, for the virus of duplicating and be packaged into of amplicon plasmid provides trans albumen, do not contain helper virus in the amplicon virus liquid that this mode produces, but owing to the lower titre of amplicon virus that makes of efficient of transfection is very low, and production all will be carried out large-scale many plasmid co-transfections at every turn, thereby is difficult to obtain the amplicon virus vector of clinical experiment aequum.The present invention adopts the Cre-loxP system with the genomic packaging signal pac excision of recombinant virus rHSV/LaL-MtCre, viral genome is reproducible and the viral various early, middle and late phase albumen of expression in cell, and the generation capsomer, but the helper virus genome can't be assembled into capsomer.Thereby when guaranteeing that the amplicon plasmid has obtained the viral protein of q.s, reduce the generation of filial generation helper virus.The amplicon virus that obtains of this method can go down to posterity on cell and increase under the condition of replenishing helper virus simultaneously, and it needs transfection repeatedly to compare with above-mentioned clay method or artificial cell karyomit(e) method, has the advantage of scale operation.

2) as the helper virus of adeno-associated virus (AAV) (AAV) carrier.The AAV carrier is because of not pathogenic to the people, but security is good, the characteristics such as long-term expression that can infect polytype cell mediate foreign gene, is one of present comparatively ideal gene therapy vector.AAV duplicates the help that needs helper virus, usually adenovirus or HSV-1 can be used as the helper virus of AAV, rep and the cap gene of AAV are inserted in the HSV-1 genome, and the reorganization HSV-1 that obtains brings up to 10 as the successful generation efficient with the AAV carrier of packing that helper virus is used for the AAV carrier ^3-4More than the TU/cell, but follow the generations of a large amount of helper viruses in the production process, make the production process of product and the final security of preparation influenced.This may become the hidden danger of AAV carrier in clinical application.In rHSV/LaL-MtCre of the present invention, insert rep and cap, auxiliary package AAV carrier under the condition of zine ion is being arranged, the helper virus genome duplicates in cell and expresses various trans albumen and give duplicating and packing of AAV carrier, and the genome of helper virus oneself is because packaging signal is cut, do not produce the filial generation helper virus, therefore the helper virus content in the AAV carrier that obtains significantly reduces, and has improved the reliability of the abundant deactivation of helper virus in the AAV carrier.

(2) as transgenosis and Vectors in Gene Therapy

The rHSV/LaL-MtCre that the present invention proposes can be used as transgenosis and Vectors in Gene Therapy, by regulating the concentration of the zine ion in the environment, can reach the control of duplicating and increasing of the rHSV/LaL-MtCre in pair cell or the human body, DNA efficient shift or gene therapy in as carrier.If further with some genetically deficient in the rHSV/LaL-MtCre genome or add some target gene, make the propagation in tumour cell of virus-specific and destroy tumour cell, be expected to be used for the gene therapy of diseases such as malignant tumour.

(3) be used for the preparation of HSV-1 ghost

Empty particle all has boundless application prospect in the molecular biology fundamental research of virus immunity, transgenosis and gene therapy, virus and cell.The rHSV/LaL-MtCre that the present invention proposes produces a large amount of virions that do not contain viral DNA under the zine ion condition, can obtain a large amount of HSV-1 ghosts after purified.

Except that HSV-1, the present invention also can be used for other Alphaherpesvirinae virus, as the transformation of people's herpes simplex types 2 virus (HSV-2) etc.Except that hsv, the present invention can be used for the helper virus of the amplicon vector of other simplexvirus, such as amplicon vector (Camp et al as Marek ' the s disease virus (MDV) of Alphaherpesvirinae, J Virol 65 (11), 1991, helper virus 6320-6324).

The primeval life material of using among the present invention

SetC clay system: wherein comprise five clay: cos6, cos28, cos14, cos56 and cos48 (Cunningham, C.and A.J.Davison 1993, Virology 197:116-124);

Cos6 Δ a, cos48 Δ a: this chamber makes up.On the basis of cos6 and cos48, with Rec Δ auxiliary restriction endonuclease patterning method (Fraefel, C., Song, S., Geller, A.I., 1996, Journal of Virology, 70,7190-7197) removed the packaging signal pac of HSV-1 respectively;

The pBS246 plasmid: GIBCOBRL company commodity plasmid provides two loxP sequences of arranging in the same way;

PMC-Cre: express the Cre recombinase protein (Hua Gu 1993, Cell 73:1155-1164) that contains nuclear localization signal (NL signal);

PCMVHyTK, be tgCMV-HyTK, (Lupton SD., Brunton, LL., Kalberg VA.and Overell RW.Dominantpositive and negative selection using a hygromycin phosphotransferase-thymidine kinase fusion gene.Mol.CellBiol.1991,11:3374-3378)

PKS-Mt:pKS, promptly pBluescriptKS is Invitrogen company product; And will be built into pKS-Mt among the Mt promotor insertion pBluescriptKS, wherein Mt promotor source is a lot, such as: plasmid pMK is disclosed in Palmiter, et al., Cell, 29:7 01-10 (1982);

P3zf-pac: this chamber makes up, and contains the packaging signal of HSV-1, be disclosed in " viral journal)) June in 1999 the 2nd phases 15 volume " structure of herpes simplex virus I-type amplicon vector)) author: Wu Xiaobing, Dong XiaoYan, Hou Yunde etc.;

PBluescriptKS (+): Stratagene company, Invitrogen company commodity plasmid;

PcDNA2.1:lnvitrogene company commodity plasmid;

PAV53: provide by this chamber doctor Wu Zhijian:

PHSV1-LacZ: make up by this chamber doctor Wu Xiaobing.

Description of drawings

The building process of cos56/loxP-pac-loxP clay of the present invention is as follows:

As shown in Figure 2.With EcoRI and BamHI double digestion p3zf-pac, reclaim the pac sequence of 1.7kb, insert in the corresponding site of pBluescript KS (+) (Stratagene company), become pKS-pac, with the pac fragment on Hind3 and the BamH1 cutting-out pKS-pac, insert in the corresponding points of pBS246 (Gibco company), become pBS246-pac, with Pst1 single endonuclease digestion pAV53, reclaim the small segment of about 1kb with an Xbal restriction enzyme site, insert the corresponding site of pcDNA2.1, screen the Xbal site nearer clone apart on this Xbal site and the pcDNA2.1, become pcDNA2.1-Xbal, cut pBS246-pac with the Not1 enzyme, reclaim the pac fragment, insert in the corresponding site of pcDNA2.1-Xbal, become pcDNA2.1-pac, cut pcDNA2.1-pac with the Xbal enzyme again, reclaim the pac fragment, insert in the Xbal site of cos56, become cos56/loxP-pac-loxP.

The building process of cos6 Δ a-MtCre clay of the present invention is as follows:

As shown in Figure 3.With XhoI and SmaI double digestion pCMVHyTK, reclaim the band plasmid skeleton of about 2.0kb and the fragment of polyA, be connected with the Mt fragment of the 800bp of SmaI double digestion pKS-Mt with XhoI, obtain pMtpolyA.The MluI enzyme is cut pMC-Cre, reclaims the cre gene of 1.1kb, mends flat back and inserts in the SmaI site of pMtpolyA, screening cre direction and the consistent plasmid of Mt promotor direction, called after pMt-Cre.With XhoI and NotI double digestion pMt-Cre, reclaim the Mt-Cre fragment of 2.1kb, load onto the XbaI joint after, insert in the XbaI site of cos6 Δ a, obtain cos6 Δ a-MtCre.

Clay cos56/loxP-pac-loxP, cos6 Δ a-MtCre that the present invention proposes are stored in intestinal bacteria (E.coli.) DH5 α strain (MAXEfficiency DH5 α respectively, GIBCO#18258-012) in, 37 ℃ of cultivations of going down to posterity in the LB substratum that contains 50-100 μ g/ml penbritin.Bacterial classification called after cos56/loxP-pac-loxP/DH5 α (the China Committee for Culture Collection of Microorganisms common micro-organisms center of containing the cos56/loxP-pac-loxP clay, deposit number: 0414), the bacterial classification called after cos6 Δ a-MtCre/DH5 α (China Committee for Culture Collection of Microorganisms common micro-organisms center, the deposit number: 0529) that contain cos6 Δ a-MtCre clay.

Embodiment

Below implement preparation and the purposes of reorganization helper virus rHSV/LaL-MtCre of the present invention have been done detailed description, but do not mean that restriction content of the present invention.

The generation of embodiment 1:rHSV/LaL-MtCre recombinant virus, go down to posterity and preserve: cos6 Δ a-MtCre, cos56-loxP-pac-loxP, cos28, cos14, five clay isoconcentrations of cos48 Δ a mix, and after cutting with the PacI enzyme, use phenol, phenol respectively: chloroform, chloroform extracting, behind the alcohol precipitation, use dissolved in distilled water DNA.Get an amount of DNA, with LipofectAMINE (GIBCO company) transfection BHK-21 cell.Picking viral plaque after 48 hours.Through behind the double plaque purifying, obtain the rHSV/LaL-MtCre recombinant virus.

The rHSV/LaL-MtCre recombinant virus converges on the BHK-21 cell of sheet behind 37 ℃ of absorption 1h about 80% with MOI=0.1, adds to contain 10% foetal calf serum nutrient solution in right amount and supported about 48 hours for 1640,37 ℃, to pathology fully.Cell and supernatant are collected in the lump, multigelation three times, the centrifugal 5min of 1500rpm collects supernatant, and the mensuration titre also is kept in-70 ℃ of refrigerators.

The Function detection of embodiment 2:rHSV/LaL-MtCre

With rHSV/LaL-MtCre and the viral HSV1-lacZ (Wu Xiaobing etc. of contrast, the virus journal, 1998,14:359-364) infect respectively and contain the BHK-21 cell of cultivating in 1640 substratum of 200 μ M zine ions (ZnCl2) (MOI=0.1), behind the absorption 1h at common 1640 substratum, the virus that flush away does not adsorb, continue to receive poison, multigelation 3 times, 1 behind the cultivation 48h with 1640 substratum that contain 10% foetal calf serum, 500rpm centrifuging and taking supernatant is measured virus titer respectively with the plaque counting process.The result shows that the titre of rHSV/LaL-MtCre in containing 1640 substratum of zine ion do not have considerable change than low two orders of magnitude of the titre in common 1640 substratum that do not contain zine ion and contrast viral HSV1-1acZ titre in above-mentioned two kinds of substratum.Illustrate that zine ion can effectively activate the expression of the Cre on the rHSV/LaL-MtCre, thereby significantly reduce the generation of progeny virus.

Embodiment 3:rHSV/LaL-MtCre is as the Function detection of helper virus to amplicon virus packing

According to the GIBCO BRL LipofectAMINE of company product description, with amplicon plasmid pHSV-LacZ transfection BHK-21 cell, behind the 12h, this cell equivalent is divided into two, and adding final concentration in the substratum of a copy of it is the zine ion of 200 μ M, after another part do not add zine ion .24 hour, infect with MOI=0.5 with rHSV/LaL-MtCre virus respectively, room temperature absorption 1h washes cell 2 times with the serum-free RPMI-1640, with containing 37 ℃ of cultivations of 10% foetal calf serum RPMI-1640.Continue to be cultured to the complete pathology of cell (about 2-3 days).Cell and supernatant are collected in the lump, multigelation 3 times, the centrifugal 5min of 1500rpm collects supernatant.

The titre of amplicon virus and helper virus detects: the viral supernatant that will collect from above-mentioned cell respectively is with 10 times of doubling dilutions, infect the BHK-21 cell, behind the absorption 1h, the virus that flush away does not adsorb, 37 ℃ are cultured to and viral plaque occurs, wash cell 3 times with PBS liquid, with the fixing 5min of 4 ℃ of stationary liquids (the PBS liquid that contains 2% formaldehyde), wash cell 3 times with PBS liquid again, add detection liquid and (contain 1mg/ml X-gal, 2mmolMgCl ₂, the 5mmol Tripotassium iron hexacyanide, the PBS liquid of 5mmol yellow prussiate of potash) and cover cell, 37 ℃ of effect 6h, microscopically dye blue cytopathy spot (malicious spot) and count dying Lan Hewei respectively.The result shows that the titre of amplicon virus (blue malicious spot) reaches 10 in the virus of the cell generation that contains zine ion ³Pfu/ml, the titre of rHSV/LaL-MtCre (not dying blue malicious spot) is less than 10 ⁵Pfu/ml; And the titre of amplicon virus has only 10 in the virus that the BHK-21 cell that does not contain zine ion produces ⁶Pfu/ml, the titre of rHSV/LaL-MtCre is 10 ⁷Pfu/ml.This result proves that the present invention can effectively reduce the content of helper virus in the amplicon virus, significantly increases the titre of amplicon virus simultaneously.

Claims

1. a reorganization HSV virus is characterized in that, has removed packaging signal pac in the HSV-1 genome of described reorganization HSV virus, and has inserted a packaging signal loxP-pac-loxP who comprises two loxP that arrange in the same way; Inserted a recombinase Cre gene that has inducible promoters in the HSV-1 genome of described reorganization HSV virus.

2. claim 1 reorganization HSV virus, wherein inducible promoters is a metallothionein promoter, this reorganization HSV viral nomenclature is rHSV-1/LaL-MtCre.

3. reorganization HSV virus according to claim 1, wherein inducible promoters is a bacterium tsiklomitsin promotor.

4. the arbitrary described reorganization HSV virus of claim 1-3 is as the purposes of helper virus.

5. the arbitrary described reorganization HSV virus of claim 1-3 is used for the purposes of the packing of HSV-1 amplicon vector as helper virus.

6. the arbitrary described reorganization HSV virus of claim 1-3 is used for the purposes of transgenosis as helper virus.

7. the arbitrary described reorganization HSV of claim 1-3 virus is used for not containing the purposes of the empty particulate preparation of hsv of DNA.